The aim of this Program Project is to discover the function of most genes in the Dictyostelium genome. In Project II we utilize concepts and methods that are centered on gene expression analysis to serve this goal. In previous years we have discovered that transcriptional profiling is a robust method for phenotypic analysis of Dictyostelium mutants and a useful means of assigning function to genes. Here we propose to study the networks that regulate gene expression during development while applying Next Generation sequencing technologies to the task. First, we will establish a 'reference transcriptome' for direct analysis of transcript abundance in the developing wild type, examining both mRNAs and non-coding RNAs. This analysis of the transcriptome will also serve as a reference for comparison with mutant strains in this project and for the entire community. Next, we will test which regulators are responsible for the dramatic transcriptional changes that accompany Dictyostelium development. We hypothesize that evolutionarily conserved transcription factors are likely to be master regulators of developmental gene expression. We will test that hypothesis by mutating the most conserved transcription factor genes and analyzing the resulting developmental phenotypes. Once we identify the transcription factors necessary for developmental progression we will use RNA sequencing (RNA-seq) and Chromatin Immunoprecipitation (ChlP-seq) to identify genes that are directly regulated by these transcription factors. Finally, we will analyze many mutants from Project 1 by transcriptional profiling, with an emphasis on mutants in genes that participate in bacterial recognition. We will intersect the transcriptional data with the fitness and the physiological data provided by Project 1 to find links between gene function and patterns of gene expression. The data we produce will also be used by Project 111 for extracting information about the genetic networks that coordinate the functions of individual genes in Dictyostelium development.